摘要
目的利用高压静脉注射转染法经小鼠尾静脉转染蛋白激酶B(AKT)、β联蛋白(β-catenin)及睡美人转座子质粒建立伴脂肪肝的炎癌转化小鼠肝癌模型并对该模型进行鉴定。方法选取6~8周龄雄性C57BL/6小鼠,通过高压静脉注射转染法在短时间内(5~9 s)经尾静脉注射含目的DNA的等渗生理溶液(20μg myrAKT、20μgΔN90-β-catenin和4μg Sleeping Beauty transposase溶于2 mL生理盐水),对照组采用相同方法注射对照质粒。于建模8、12、16周后,处死小鼠并收集血清及肝组织,提取RNA。HE染色检测肝脏组织结构及炎性细胞浸润程度;油红O染色检测肝细胞内脂质沉积情况;天狼星红和Masson染色检测肝组织纤维化程度;免疫组织化学染色检测肝组织KI-67抗原(ki67)表达显示细胞增殖活性、检测CD31表达显示微血管密度及分布情况;实时荧光定量PCR检测肝组织肿瘤标志物甲胎蛋白(AFP)的mRNA表达;流式细胞术检测肝组织内各免疫细胞亚群数量变化。结果小鼠经高压静脉注射转染法建模16周后,实验组小鼠成瘤率100%(20/20)。与对照组相比,实验组小鼠体质量增长缓慢,肝质量显著增加。HE染色结果显示,大量肝细胞出现脂肪变,炎性细胞浸润增多,肝癌组织内肿瘤细胞为类圆形或不规则形,核质比失调,异型性显著,呈团块状分布;ki67阳性细胞数增加。血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、γ谷氨酰转肽酶(γ-GT)、碱性磷酸酶(ALP)水平显著升高,AFP mRNA表达显著上升,CD31表达检测显示微血管密度增加且结构紊乱。流式细胞术分析肝脏中大量炎性细胞浸润,且以髓系及淋巴系为主。结论通过高压静脉注射转染法在肝细胞中激活AKT和β-catenin成功建立了伴发脂肪肝的炎癌转化小鼠肝癌模型,且方法简便,诱导时间短,成瘤率高,重复性强。
Objective To establish and evaluate a murine lipogenic inflammation-mediated hepatocarcinoma model with hydrodynamic transfection of protein kinase B(AKT),β-catenin,and Sleeping Beauty transposon plasmids into hepatocytes through the tail vein.Methods Isotonic physiological solution(20μg myrAKT,20μgΔN90-β-catenin and 4μg Sleeping Beauty transposase were dissolved in 2 mL of normal saline)was injected into C57BL/6 male mice aged 6-8 weeks via the tail vein in a short period of time(5-9 s)by hydrodynamic transfection,while the control plasmid was injected with the same method.Mice were then sacrificed 8,12,or 16 weeks after the injection of these plasmids,and their serum and liver tissues were collected.RNA samples were extracted for further analysis.HE staining was used to detect liver tissue structure and inflammatory cell infiltration;oil red O staining was used to examine lipid deposition in liver cells;sirius red and Masson staining were used to identify collagen deposition in liver tissues;immunohistochemistry was performed to evaluate the proliferation abilities of the cells and microvascular density and distribution;real-time quantitative PCR was used to reflect the expression of tumor-associated genes;flow cytometry was used for immune cell phenotype identification.Results Sixteen weeks after hydrodynamic transfection,100%(20/20)mice developed liver tumors in the experimental group.Compared with the control group,the body mass of mice in the experimental group increased slowly and liver mass increased significantly.HE staining showed that a large number of hepatocytes appeared to be steatosis,inflammatory cell infiltration increased,liver tumor cells were round or irregular-shaped,the cytoplasmic ratio was imbalanced,tumor cell atypia was obvious and tumor cells were distributed in clustering;ki67 positive cells were enhanced.Serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),γ-glutamyl transpeptidase(γ-GT),and alkaline phosphatase(ALP)levels remarkably increased.AFP mRNA also significantly increased in liver tissue.CD31 staining showed increased vessel density and vessel structure disorganization.A large number of inflammatory cells,especially myeloid and lymphoid cells,were infiltrated into the liver tissue.Conclusion Murine lipogenic inflammation-mediated hepatocarcinoma model that is caused by non-resolving inflammation was successfully established by hydrodynamic transfection with AKT andβ-catenin plasmids.This method is easier performed and reproducible,the induction time is relatively short with a high rate of tumor formation.
作者
卢羿同
杨嘉蒙
赵俊龙
秦鸿雁
LU Yitong;YANG Jiameng;ZHAO Junlong;QIN Hongyan(Department of Medical Genetics and Developmental Biology,School of Basic Medicine,Air Force Medical University,Xi’an 710032,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2020年第2期122-129,共8页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金面上项目(31570878,81872265)。